Genes from the cytochrome P450 (CYP) superfamily encode a large and diverse group of mixed-function oxygenases whose genetic variation is known to be medically significant. P450 proteins in the CYP2 family are among the most important enzymes in mammals for the metabolism of a wide range of environmental and ingested compounds, including many drugs, and in many endogenous processes. This project has the long-term goals of achieving a better understanding of the evolutionary processes that have created the CYP2 gene family, and of assessing the suitability of several animal models for some specific CYP2 enzymes. Analyses of the CYP2 clusters in human and mouse have provided important information regarding medically significant genes in the group and have allowed us to infer some of the mechanisms behind the evolution of the genes. Elucidating the structure, relationships, and regulation of these genes in the rat, an important model system for P450 enzymes, will permit us to correlate biomedical studies across species, while information on the corresponding genes in two additional species, the cat and the opossum, will help us to understand evolutionary processes in gene clusters. These three species represent three different levels of relatedness to the human and mouse, and all three are employed as animal medical models. A detailed physical map of the CYP2 gene cluster will be created for each species by isolating and restriction mapping genomic clones from the appropriate BAC libraries. Genes will be localized on the map by hybridization with specific probes. Individual genes will be conclusively identified, and previously unknown loci will be sequenced and analyzed. The sequences and restriction maps will be correlated with all information available from the rat and cat genome projects. Full-length genes not associated with known transcripts will be tested to determine whether they are expressed. The cluster maps from all five mammals (human, mouse, rat, cat opossum) will be compared, orthologous genes will be aligned, regulatory elements will be identified, and the events that have shaped this gene family group during mammalian evolution will be inferred.